Team:OUC-China
Background
Nowadays, strain quality has become a major factor limiting the yield of fermentation industry. The engineering bacteria degenerate into wild-type due to mutation or some unknown endogenous regulation. They have more growth advantages than the production engineering bacteria and gradually occupy resources to become the dominant species. And the extraction process of the products by breaking the engineering bacteria is very complicated. We plan to design a platform that automatically regulates the production and screening of strains to extract product simply to ensure high-quality strains and improve the fermentation yield.
Quorum sensing
Quorum sensing
design
We use cytokinin-mediated quorum sensing circuit from Arabidopsis thaliana makes our engineered microorganisms only reaches a certain population density will start fermentation process, used for decoupling competition between cell growth and fermentation.
When the signal molecule IP reaches a certain concentration, it activates the SSRE promoter and increases the expression of production gene controlled by SSRE promoter.Therefore, when the number of engineered bacteria in the fermentation tank is small, the expression of the production genes will remain very low, which makes the engineered bacteria have higher competitiveness to the wild bacteria.
Test
In our experiment,to better characterize SSRE promoter and quorum sensing circuit,we put GFP gene at the downstream of SSRE promoter then detect them in Aureobasidium melanogenum.
Learn
We also construct a gene circuit to help maintain or increase the copies of production gene compared with common production bacteria,which brings out a contradiction between increase of expression and regulatory of expression.We built a system biology model to simulate the interaction between components in the quorum sensing system.It can prove the feasibility of our quorum sensing system, because although the number of plasmid copies is gradually increasing, the expression of SSRE promoter does not increase significantly, which means that the SSRE promoter can indeed play a role in regulating the start of fermentation at the early stage of fermentation and tactfully handle the contradiction with the number of copies at the same time.
To have a great insight for quorum sensing,we simulated the behavior of three proteins before and after phosphorylated in quorum sensing system.
Design
To add more parts with various of dynamic ranges, we simulated the expression of different subtypes SSRE promoters based on our learning result,which will provide users of our platform with more choices.Because SSRE promoters of different subtypes have different dynamic ranges, different SSRE promoters can be selected according to the simulation results to the needs of different fermenter sizes.Then the expression intensity of different SSRE promoter was simulated in our system
RNA transporter and hardware
We decided to design a transport aptamer which can target membrane structures so that they can play a role in cells as transporters. But because of the epidemic situation, our collaborative team did not have time to help us with in vitro transport experiments using exosomes. Our hardware can adjust the outflow rate of the fermentation liquid through the regulation of the microvalve controller, thus ensuring that the transport RNA will only have minimal probability of transporting the product back into the cell again.And the transport aptamer works along with the hardware, so we decided to analyze the performance of both together.
Test
We obtaines some aptamers and measured the aptamers binding abilities to the product molecule . In other words, we got aptamers with different Kd values.
Learn
We learned the sequence of these aptamers and the ability to specifically bind the product molecules.Because Disp is a platform, all of our parts need to be designed for different fermentation situations, thus requiring aptamers with different Kd values.
Design
We used molecular docking and the relationship between the sequence and the Kd value to add more aptamers with different Kd values. These aptamers were used for the modification and were designed as different transport aptamers to meet the different fermentation conditions. Then we used aptamers with different Kd value to construct transport RNA that can bind to membrane structure and specifically to the product by simply replacing the product binding aptamer sequence in RNA10,then transport the product along the concentration gradient.
Test
Using the above transport RNA constructed with different Kd value aptamers, we simulated different cases of transport aptamers through simulation, which is sufficient to prove the feasibility of our hardware.
Kill switch
Design
To enable the engineered microorganism to initiate suicide at low yields, we designed kill switch whose aptamer faction binding to the product would activate ribozyme activity, causing the designed mRNA to self-cleavage and preventing YopE (kill protein)expression.
Build
Firstly, the model was constructed to analyze the content of products (inside and outside cells) in the whole system after a period of fermentation and we could see that the level of the products was stable after a period of fermentation, which also verified the feasibility of the riboswitch.
Because under normal conditions, the level of intracellular products is basically unchanged, and if some mutations or other factors cause the production to decline, the riboswitch will work. This also provided confidence for us to further test the function of the kill switch.
Test
We examined the performance of the suicide switch by replacing upstream of the ribozyme riboswitch to the eGFP gene. However, the experimental data of the riboswitch is not ideal due to detection equipment failure, so we used simulation modeling to verify its feasibility. We constructed a systems biology model, which first changed the yield in the system, and we can see that with the higher production rate, the YopE protein expression decreased significantly by figure6A, and its production rate increased significantly. After continuing the exploration, we found that there is another element in the system that can affect the yield change in the system, which is the ability of the riboswitch to bind to the ligand. By changing the binding capacity of the aptamer, it can be seen that changing the binding capacity of the aptamer can effectively inhibit the expression of YopE protein, thus reducing the manslaughter phenomenon.
Overall test
Because we can't use the large fermentation tank used in the company's production, it is difficult for us to know whether the platform we designed has the function of successfully enabling the engineering bacteria to automatically adjust the production and growth continuously ferment to increase production.After adjusting the components of each component to achieve the most effective, it is obvious that engineered bacteria with CHIP introduced have higher production and have better fermentation effect under the same number of engineering bacteria by using cellular automata to conduct integrity analysis
